Many plant species are known to undergo masting, where individuals within a species all produce a large amount of seed in a single year, with a sharp drop off in reproduction in the following years. Masting may be beneficial to plants by suppressing seed predator populations in low seed production years, allowing for seeds to escape predation in high seed production years. Masting is likely associated with climate, although this is poorly understood. In this study, I will use herbarium specimens to tackle questions related to masting in five plant species: Tsuga heterophylla, Pseudotsuga menziesii, Thuja plicata, Rubus spectabilis, and Rubus parviflorus. Specifically, I will note the number of cones, seeds or berries on herbarium specimens, as well as specimen size and the year it was collected. I will use these data to 1) examine patterns of masting in focal species as compared to field data from Mount Rainier. I hypothesize that herbarium specimens will show a similar pattern of masting as field data, with trees showing stronger patterns than shrubs. Next, I will use these data to 2) compare masting patterns to annual climate. I hypothesize that warm temperatures lead to masting the following year, and that masting has increased in frequency. Finally, I will 3) assess the relationship between masting and bird species that eat seeds and berries (e.g. grouse, jays), to determine whether masting influences population dynamics of higher trophic levels. I will do so by comparing masting patterns with bird count data from the Audobon Society. I hypothesize that population sizes of birds that rely on seeds and berries will be greater in mast years. This study provides additional information for how we might expect entire ecosystems to be affected by climate change, including resource distribution and population health.

The tumor microenvironment (TME) dictates the outcome of many immuno-oncology therapies for solid tumors. To better understand the dynamic milieu of the TME, new multiplexed, spatially-resolved histologic techniques are being developed. A key limitation to evolving these techniques is identifying specific and selective antibodies that perform well in an immunohistochemistry (IHC) platform. The over-arching goal of this project is to develop a flexible, high-throughput platform to empirically test the IHC staining characteristics of antibodies in formalin-fixed paraffin-embedded (FFPE) tissues with improved throughput and lower test volumes. Essential elements in our platform design include: 1) flexibility to test a variety of biologic materials, which is dependent on the test antigen, 2) compatibility with manual and semi-automatic tissue microarray (TMA) builder, 3) easy use for pathologic assessment, and 4) future compatibility with fully-automated tissue staining instrumentation. Using computer-aided design software, and a stereolithography printer, we prototyped a guide template to build recipient TMA FFPE blocks. To screen for a variety of antigens in TMA "cores", we prepared cell lines, and acquired mouse tumor xenografts and human tissues. Standard IHC techniques were used to screen hybridoma supernatants generated from mouse immunizations. We have designed and tested six different multi-well slide-based IHC screening platforms. The current format consists of a six by four array of 2 mm cores. We have screened antibodies from two different mouse hybridoma campaigns. Thus far, we have identified candidate IHC antibodies for exogenous epitopes to identify chimeric antigen receptor T cells used to treat solid tumors, and a fusion protein hypothesized to be an oncogene in pediatric liver cancer. This project developed a single-slide antibody screening prototype for IHC. The device offers the flexibility to test multitude of tissues, and is built with design considerations for future automated tissue staining compatibility.

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